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Leptospirosis is a zoonosis caused by Leptospira bacteria present in the urine of mammals. Leptospira is able to survive in soils and can be resuspended during rain events. Here, we analyzed the pathogenic Leptospira concentration as a function of hydrological variables in a leptospirosis hot spot. A total of 226 samples were collected at the outlet of a 3 km 2 watershed degraded by ungulate mammals (deer and feral pigs) and rats which are reservoirs for leptospirosis. Water samples collected at the beginning of a rain event following a dry period contained high concentrations of pathogenic Leptospira. The concentration was generally correlated with the water level and the suspended matter concentration (SMC) during the main flood event. A secondary peak of pathogenic Leptospira was sometimes detected after the main flood and in slightly turbid waters. Lastly, the pathogenic Leptospira concentration was extremely high at the end of a wet season. The pathogenic Leptospira concentrations could not be explained by a linear combination of hydrological variables (e.g. the rainfall, water level, SMC and soil moisture). However, nonlinear machine learning models of rainfall data only provided a fair fit to the observations and explained 75 % of the variance in the log 10 -transformed pathogenic Leptospira concentration. A comparison of identical machine learning models for the water level, SMC and pathogenic Leptospira concentration showed that the residual error in the Leptospira concentration was due to not only the small dataset but also the intrinsic characteristics of the signal. Our results support the hypothesis whereby pathogenic Leptospira survive at different depths in soils and superficial river sediments (depending on their water saturation) and are transferred to surface water during erosion. These results might help to refine leptospirosis warnings given to the local population. Future research should be focused on larger watersheds in more densely populated areas., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in relation to the Ms. entitled: Rainfall-driven pathogenic Leptospira resuspension in a leptospirosis hotspot, submitted to Science of the Total Environment., (Copyright © 2023. Published by Elsevier B.V.)

Mostly studied as a zoonosis, leptospirosis is also an environment-borne infection and most human cases originate from soil or water contaminations. Yet, only few studies have been interested in the survival of pathogenic Leptospira in freshwater. In this study, water microcosms were designed to evaluate the survival and virulence of Leptospira spp. for 2 years. Four commercial bottled drinking waters and a non-ionized water, all previously filter-sterilized, were studied. Either one of two Leptospira interrogans strains, one Leptospira borgpetersenii strain, or a saprophytic Leptospira biflexa was inoculated in these waters under nutrient-deprived conditions. Molecular, microscopic and cultural approaches were used to study Leptospira survival. Direct virulence of the pathogens was assessed using animal challenge without re-culturing. Our results confirmed the capacity of pathogenic Leptospira to survive for more than a year in water. In addition, we showed the ability of L. interrogans in nutrient-deprived conditions to directly cause systemic infection in susceptible animals. To our knowledge, this is the first report of direct infection of a susceptible host with Leptospira following a long starvation and survival period in nutrient-deprived water. Our results also suggest that Leptospira turned into a physiological "survival" state in harsh freshwater conditions. These data are of prime importance considering that freshwater is a major source of Leptospira infections. Environmental survival and virulence of pathogenic Leptospira spp. are becoming a crucial challenge to determine the environmental risk and adopt relevant prevention and control strategies.

Background: Leptospirosis, caused by pathogenic Leptospira, is a zoonosis of global distribution. This infectious disease is mainly transmitted by indirect exposure to urine of asymptomatic animals via the environment. As human cases generally occur after heavy rain, an emerging hypothesis suggests that rainfall re-suspend leptospires together with soil particles. Bacteria are then carried to surface water, where humans get exposed. It is currently assumed that pathogenic leptospires can survive in the environment but do not multiply. However, little is known on their capacity to survive in a soil and freshwater environment., Methods: We conducted a systematic review on Leptospira and leptospirosis in the environment in order to collect current knowledge on the lifestyle of Leptospira in soil and water. In total, 86 scientific articles retrieved from online databases or institutional libraries were included in this study., Principals Findings/significance: This work identified evidence of survival of Leptospira in the environment but major gaps remain about the survival of virulent species associated with human and animal diseases. Studies providing quantitative data on Leptospira in soil and water are a very recent trend, but must be interpreted with caution because of the uncertainty in the species identification. Several studies mentioned the presence of Leptospira in soils more frequently than in waters, supporting the hypothesis of the soil habitat and dispersion of Leptospira with re-suspended soil particles during heavy rain. In a near future, the growing use of high throughput sequencing will offer new opportunities to improve our understanding of the habitat of Leptospira in the environment. This better insight into the risk of leptospirosis will allow implementing efficient control measures and prevention for the human and animal populations exposed., Competing Interests: The authors have declared that no competing interests exist.

Medical microbiology has used phenotypical and metabolic criteria to identify bacterial pathogens for decades. However, no such criteria have been applied to identify leptospires at the species level. In the recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) has emerged as new tool for the identification of bacterial species in the medical microbiology laboratory. This technology has rapidly gained more and more popularity. Actually, this technique is sensitive and economic, saving both labor and bench costs, but also rapid, significantly reducing turnaround time from isolation to identification. MALDI-ToF MS provides an unprecedented tool for the rapid identification of Leptospira at the species level.

Leptospires, the etiological agents of leptospirosis, are fastidious slow-growing organisms. Here we describe the isolation and routine maintenance of leptospires from clinical (blood, urine, or tissue) and environmental (water or soil) samples. Using combinations of filtration, agar plating, and selective agents, leptospires can be isolated in pure cultures even from complex contaminated sources in standard EMJH culture medium.

The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species., Competing Interests: The authors have declared that no competing interests exist.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral 'pathogens' cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.